Neutrophil extracellular traps (NETs) are released by neutrophils in a process called NETosis, a form of cell death triggered by various agents like phorbol myristate acetate (PMA), bacterial lipopolysaccharide (LPS), and bacteria such as *Staphylococcus aureus* and *Pseudomonas aeruginosa*. These agonists activate NADPH oxidase (NOX), generating ROS and activating mitogen-activated protein kinases. Previous research established that genome-wide transcriptional firing, initiated by kinase cascades, is necessary for chromatin decondensation and NETosis. However, the role of DNA repair in NETosis remained unclear. While ROS is essential for NETosis (inhibition of ROS production completely inhibits NETosis), the mechanism by which ROS executes NETosis was unknown. The authors hypothesized that ROS induces DNA damage, and the subsequent repair of this damage via DNA repair pathways leads to chromatin decondensation and NETosis. ROS can oxidize DNA bases (e.g., converting guanine to 8-oxoguanine), and when transcription machinery stalls at damaged sites, the repair machinery assembles, opening the chromatin for repair. This process involves enzymes like 8-Oxoguanine glycosylase (OGG1), PCNA, apurinic/apyrimidinic endonuclease (APE1), poly(ADP-ribose) polymerase (PARP), DNA ligase, and DNA repair polymerases (pol) β and δ. The study focused on investigating the importance of ROS-mediated DNA damage and each key step of the DNA repair pathway in driving chromatin decondensation and NET formation.

The literature review section extensively cites previous research on NETosis, highlighting the role of ROS and NADPH oxidase. It references studies demonstrating the necessity of ROS for NETosis and the involvement of MAPK pathways. Furthermore, it mentions prior work showing the importance of genome-wide transcriptional firing in driving NETosis but notes the lack of understanding concerning the role of DNA repair in this process. The existing literature provides the groundwork for the authors’ hypothesis that ROS-induced DNA damage and subsequent repair are key drivers of NETosis. The review emphasizes the known components of base excision repair (BER) and nucleotide excision repair (NER) pathways, laying the foundation for the experimental design that focuses on the role of key enzymes in these pathways in NETosis.

The study utilized human neutrophils isolated from peripheral blood of healthy donors. NETosis was induced using PMA and LPS, and the extent of DNA damage was assessed by measuring 8-oxoG levels using immunofluorescence confocal microscopy and an in-cell ELISA. The subcellular localization of PCNA was tracked using confocal microscopy. To determine the importance of different steps in the DNA repair pathway, the authors employed SYTOX Green plate reader assays to measure NETosis in the presence of various inhibitors targeting APE1, PARP1, DNA ligase, PCNA, and polymerases β/δ. These inhibitor studies were further confirmed using siRNA-mediated knockdown of APE1 and PARP1 in differentiated HL-60 cells. The effect of DNA repair inhibitors on bacteria-induced NETosis was also tested using *Staphylococcus aureus* and *Pseudomonas aeruginosa*. Statistical analyses included one-way ANOVA, two-way ANOVA, and Student's t-tests. Detailed protocols for neutrophil isolation, SYTOX Green assays, confocal imaging, siRNA knockdown, in-cell ELISA, and statistical analyses are provided in the methods section.

The study found that PMA and LPS treatments induced significant increases in 8-oxoG levels, indicating extensive DNA damage. PCNA, usually cytoplasmic in resting neutrophils, translocated to the nucleus following NETosis induction. Inhibition of the early steps of the BER/NER pathway (APE1, PARP1, and DNA ligase) significantly suppressed NETosis induced by PMA, LPS, *S. aureus*, and *P. aeruginosa*. Conversely, inhibiting the later steps (PCNA interactions with polymerases β/δ or DNA polymerase β activity) did not significantly affect NETosis. siRNA knockdown of APE1 and PARP1 in HL-60 cells also reduced NETosis, confirming the inhibitor data. The results indicate that the initial chromatin decondensation steps of BER/NER, but not the steps after PCNA binding and DNA polymerase activity, are necessary for ROS-mediated NETosis. The observed effects were consistent across different NETosis inducers (PMA, LPS, *S. aureus*, *P. aeruginosa*).

The findings demonstrate a novel mechanism by which ROS induces NETosis. The study suggests that ROS initially triggers a genome-wide transcriptional response and simultaneously oxidizes DNA, leading to DNA damage. The subsequent activation of the DNA repair machinery, specifically the early steps involving APE1, PARP1, and DNA ligase, drives chromatin decondensation, facilitating NET release. The authors propose that the chromatin unwinding capacity of the DNA repair machinery is a key factor in NETosis. The observation that inhibiting later steps of the DNA repair pathway does not affect NETosis suggests that the initial chromatin opening is sufficient for the process. The authors discuss the multifunctional nature of APE1 and suggest that the observed effects are due to its endonuclease activity rather than its redox activity. The independence of DNA replication from NETosis is also highlighted.

This study reveals a critical role for DNA repair in ROS-mediated NETosis. The early steps of the base excision repair pathway are essential for NET formation, with the initial chromatin decondensation being sufficient for NETosis to occur. This work provides new insights into the mechanisms underlying NETosis and suggests potential therapeutic targets for NETosis-related diseases. Future research could focus on exploring the relative contributions of different DNA repair pathways and the potential of specific DNA repair pathway inhibitors in treating NETosis-associated pathologies.

The study primarily focuses on NOX-dependent NETosis inducers and may not be fully generalizable to all forms of NETosis. The use of inhibitors may have off-target effects, although the authors attempted to minimize this by using multiple inhibitors and verifying their function in separate assays. The study primarily used in vitro models which may not entirely reflect the complexity of NETosis in vivo. Future research could investigate in vivo models to validate the findings.